J. Biol. Chem., Vol. 266, Issue 23, 15016-15020, 08, 1991
Effects of a leucine analog on growth hormone processing and secretion by cultured cells
SJ McAndrew, NY Chen, B Kelder, JA Cioffi and JJ Kopchick
Department of Zoology, Ohio University, Athens 45701.
Bovine and rat growth hormones (bGH and rGH, respectively) possess signal
peptides that direct the hormone to the secretory pathway and are
proteolytically cleaved prior to secretion. Previous in vitro translation
studies indicated that incorporation of the polar leucine analog
beta-hydroxyleucine into de novo synthesized polypeptides inhibits signal
peptide function. To test the effects of this analog on GH secretion by
cultured animal cells, transfections of mouse L-cells with a bGH expression
plasmid or metabolic labeling of endogenous rGH in anterior pituitary cells
was performed in the absence or presence of beta-hydroxyleucine. Transient
expression of bGH in mouse L-cells or endogenous expression of rGH in
anterior pituitary cells resulted in an accumulation of GH in the culture
medium. Treatment with beta- hydroxyleucine resulted in a block in
secretion as evidenced by an accumulation of GHs within these cells.
Amino-terminal sequencing of the intracellular form of the
analog-substituted GHs demonstrated accurate signal peptide cleavage. In
contrast, in vitro translations of bGH RNA performed in the presence of
beta-hydroxyleucine and microsomal membranes resulted in the inhibition of
signal peptide cleavage. The results suggest that beta-hydroxyleucine can
uncouple signal peptide processing and protein secretion in cultured cells.
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